The present study aimed to evaluate the stability of Human Adenovirus type 2 (HAdV2) and Murine Norovirus 1 (MNV-1) in surface freshwater samples stored at different temperatures. For HAdV2 the stability decreased with increasing temperatures (−80 > −20 > 4 > 22 °C). The time required to reach one log reduction in viral titers (T90) was similar among all the times and temperatures by different cell-culture based methods and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The HAdV2 stability decreased with the time of storage temperature and methods employed, aside from samples stored at 22 and 4 °C which showed the lowest T90 values (50 days). For MNV-1, the samples stored at 22 and −20 °C showed higher log10 decay values, followed by 4 and −80 °C; while genome persistence was ranked as −80 > −20 > 4 > 22 °C. The T90 values were lower for samples stored at 22 °C (33 days), followed by 4, −20 and −80 °C with 111, 100 and 333 days, respectively. The results indicate that, under laboratory storage conditions, freshwater samples should be kept at 4 °C and at −80 °C for short- and long-term periods, respectively. This study provided useful information about thermal and temporal stability of the enteric viruses regarding sample storage conditions.
Surface water reservoirs are one of the most important sources of drinking water in the world. The quality of these reservoirs is constantly threatened by the direct or indirect discharge of contaminated effluents and poses health risks to water consumption as well as recreational and agricultural uses.
Many studies have reported contamination in reservoirs by different pathogens including bacteria, protozoa, and viruses (Ogorzaly et al. 2009; Rigotto et al. 2010; Gallas-Lindemann et al. 2013). Among these, contamination caused by enteric viruses is of particular concern due to high infectivity and resistance to traditional water treatment processes. Furthermore, enteric viruses are a leading cause of water-related diseases, and are capable of exhibiting a broad range of waterborne illnesses (Lysén et al. 2009; Sinclair et al. 2009).
The presence and persistence of viruses should be monitored to ensure water quality; however, extensive sampling schedules can preclude immediate analyses and require samples to be stored before further analyses. Presently, there is a dearth of information regarding the stability of enteric viruses in environmental samples under laboratory storage conditions (−80, −20, 4, 22 °C).
Although temperature is considered the principal factor affecting viral stability, other biotic and abiotic factors can contribute to the loss of viral infectivity in water samples. Such factors include the presence of endogenous micro-organisms that can decrease viral viability through predation or virucidal activity; inactivation by sunlight incidence; pH variation and salt concentration (Ward et al. 1986; Hurst 1988a; Bertrand et al. 2012). In addition, the adhesion phenomena between viruses presents in environmental water and the storage container walls must be considered regarding viral inactivation (Gassilloud & Gantzer 2005). On the other hand, phenomena such as virus aggregation to solid particles in the aqueous phase or aggregation between viruses themselves can increase virus stability, protecting from the action of inactivation factors (Gassilloud et al. 2003).
Studies evaluating enteric viral stability and inactivation in different matrices and under different conditions, usually employ robust and well-established viral models, such as human adenoviruses (serotypes 2, 5, 40 and 41), human enterovirus (poliovirus 1, coxsackievirus, echovirus), rotaviruses (RV) and murine noroviruses (MNV-1) (Raphael et al. 1985; Hurst et al. 1988b; Bae & Schwab 2008; Charles et al. 2009; Rigotto et al. 2011).
Due to the relevance for the viral infectivity and stability studies the main objective of this study was to evaluate the stability of Human Adenovirus type 2 (HAdV2) and Murine Norovirus 1 (MNV-1) in surface freshwater samples at different temperatures (including laboratory storage conditions) over time. Furthermore, samples were evaluated by a variety of cell-culture and molecular based methods to analyze the differences between infectivity and genome persistence.
HAdV2 and MNV-1 were propagated in A549 and RAW 264.7 cell lines, respectively, in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL, Life Technologies do Brazil, São Paulo, SP, Brazil) supplemented with either 10% (growth medium) or 2% (maintenance medium) of heat-inactivated fetal bovine serum (Gibco) and 100 U/mL penicillin G, 100 μg/mL streptomycin, 0.025 μg/mL amphotericin (PSA) (Gibco). RAW 264.7 cells were also supplemented with 2 mM l-glutamine, 1 M HEPES, and 1× non-essential amino acids (Sigma-Aldrich, St Louis, MO, USA). Viral stocks of HAdV2 (6.8 × 108 PFU/mL) and MNV-1 (3.5 × 106 PFU/mL) were titered by plaque assay (PA) as described below and aliquots were stored at −80 °C for further experiments.
Water matrix, storage conditions and sampling schedule
Raw freshwater from Peri Lagoon was selected as the water matrix to evaluate the thermal and temporal stability of the HAdV2 and MNV-1. This water is caught by local water treatment plant for treatment and further distribution to the local population and is also used for recreational activities during the summer season. Adenoviruses, hepatitis A virus, polyomavirus JC, and RV genomes have been detected in this aquatic environment in a previous surveillance study from our research group (Fongaro et al. 2012).
The following physicochemical parameters of the water matrix were taken in situ using a YSI-85 multiparameter probe (YSI Incorporated, Yellow Springs, OH, USA): temperature 25 °C, pH 6.09, turbidity 5.6 NTU and conductivity 67.2 μS/cm.
The viruses were spiked in separate water aliquots to reach a final concentration of 1.0 × 108 PFU/mL for HAdV2 and 2.4 × 105 PFU/mL for MNV-1. These concentrations were chosen to achieve a 99.99% reduction (4 log10) in viral titers. After spiking, the water samples were divided into 1 mL aliquots (polypropylene vials) and stored at 22, 4, −20 and −80 °C in dark conditions. The infectivity of HAdV2 was assessed via PA, flow cytometry (FC), and integrated cell culture-reverse transcription-quantitative polymerase chain reaction (ICC-RT-qPCR), at 0, 5, 10, 15, 30, 60, 120, 180 and 230 days after spiking. For MNV-1, the viral infectivity and genome stability were evaluated, respectively, by PA and RT-qPCR at 0, 5, 10, 15, 30, 60, 90, 120 and 180 days after spiking.
HAdV2 infectivity assays
The PA for infectious HAdV2 was performed as described by Cromeans et al. (2008), with minor modifications. Briefly, A549 cells monolayer were infected, in duplicate, with 0.25 mL dilutions of spiked water samples and overlaid with 0.3% bacto-agar (LGC Biotecnologia, Brazil). After incubation (6–7 days) the cells were stained with 0.4% crystal violet, 0.1% phenol and 12% ethanol diluted in water, to visualize plaques. Macroscopic plaques were counted to establish viral titers expressed as plaque-forming units per mL (PFU/mL).
The detection of infectious HAdV2 by FC was performed as described by Corrêa et al. (2012) with some modifications. A549 cells were incubated with spiked water samples for 48 h. After this period, the supernatant was removed and the cells were harvested with trypsin (2.5 mg/mL). The cells were firstly incubated with a specific monoclonal antibody (MAb8052) (Nihon Millipore™, Tokyo, Japan) followed by incubation with anti-mouse IgG conjugated to FITC (Sigma-Aldrich). The presence of fluorescent cells (infected by HAdV2) was analyzed in a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) (10,000 events per sample) and the results expressed as green fluorescent units per mL (GFU/mL) (Gueret et al. 2002).
The procedure employed in this method was based on previous studies described by Ko et al. (2003, 2005) and Fongaro et al. (2013), with some modifications. Briefly, A549 cells were infected with the water samples and after 24 h of incubation the supernatant was removed followed by direct RNA extraction from the infected cells monolayer using a commercial RNeasy-MinElute Kit (Qiagen, Valencia, CA, USA). The extracted RNA was treated with 2 U of DNase I (Sigma-Aldrich) and a reverse transcription assay was performed using random primers and Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA). The quantification of the HAdV2 genomes (hexon gene) were performed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the protocol described by Hernroth et al. (2002). The samples were analyzed in duplicate with positive (standard curve) and non-template controls included in each run.
MNV-1 stability assays
The PA for infectious MNV-1 was performed as described by Bae & Schwab (2008). RAW 264.7 cells were infected with dilutions of the spiked water samples in duplicate and overlayed with 3% of SeaPlaque agarose (Lonza, Basel, Switzerland). The cells were incubated for 36 to 48 h and stained with a solution of 0.01% of Neutral Red (Sigma-Aldrich) diluted in phosphate buffered saline (PBS). The values of infectious MNV-1 were expressed as PFU/mL.
RNA extraction and RT-qPCR for MNV-1
The RNA from the water samples spiked with MNV-1 was extracted with the QIAmp®MinElute Virus Spin Kit (Qiagen) following the manufacturer's instructions. MNV-1 quantification was based on the RT-qPCR assays described by Baert et al. (2008) using a tenfold dilution of RNA from each sample. The reactions were performed in duplicate using the StepOnePlus™ Real Time PCR System (Applied Biosystems). Positive (standard curve) and non-template controls were added in each run.
Viral decay for all assays was established by comparing viral titers at successive time points (Nt) to the initial concentration of viruses immediately after spiking water matrices (N0), following equation [log10(Nt/N0)]. A linear regression was employed to obtain the relationship between viral decay and time (R2). The slope of this line was used to establish the coefficient of the inactivation rate –k, thereby allowing us to establish the T90 value. An analysis of variance was performed in order to compare the log10 reduction values and the T90 inactivation times obtained for both viruses and in the different assays. Graphics and statistical analysis were performed using GraphPad PRISM 5.00 software (GraphPad Software, La Jolla, CA) using a P value of 0.05.
RESULTS AND DISCUSSION
Thermal and temporal HAdV2 stability
When assayed by FC, HAdV2 showed an initial reduction in viral titers (Figure 1(b) – day 5), but remained constant until 60 days after spiking (P < 0.05). Samples stored at 22 °C did not exhibit this consistency, nor was a similar decay curve observed, as in other defined temperatures. At the end of 230 days the virus inactivation by FC reached the following values: 5.13 log10 (22 °C), 3.49 log10 (4 °C), 3.44 log10 (−20 °C), and 3.43 log10 (−80 °C) (P < 0.001).
Regardless of storage temperature, HAdV2 stability showed lower reduction in viral titers within the first 60 days of analysis when analyzed by ICC-RT-qPCR (Figure 1(c)). After this period, viral stability declined (nearly consistently) until the end of the study. At 230 days, the decay values were as follows: 4.53 log10 (22 °C), 1.79 log10 (4 °C), 1.78 log10 (−20 °C), and 1.45 log10 (−80 °C), with (P < 0.01).
There are few studies evaluating the stability of infectious viruses in water matrices under environmental and laboratory storage temperature conditions. In the present work, the stability of HAdV2 varied according to assay method and storage temperature.
Overall, FC exhibited the highest reductions in viral titers over time, followed by PA and ICC-RT-qPCR, respectively. All employed methods exhibited a non-linear decay during the initial times evaluated (first 30 days for PA and FC, 10 days for ICC-RT-qPCR), which may be indicative of viral aggregation (Gassilloud & Gantzer 2005).
The interaction between the viruses and the storage container walls (adhesion phenomena) may also be considered due to the possible significant impact on the loss of the virus infectivity. Gassilloud & Gantzer (2005) found 30 times more PV1 genome adhering to the polypropylene storage container walls than in the aqueous phase, at the first 20 days of groundwater samples storage. Despite the evidence of the adhesion phenomena, polypropylene containers (most hydrophobic) appears to promote less adherence to the walls in comparison with containers composed of other materials, such as Pyrex glass and polystyrene (Ward & Winston 1985). In the present study, 1 mL polypropylene vials were used for freshwater sample storage. Usually, the occurrence of adhesion phenomena can be detected over a short period of time and may explain the initial 2 log10 reduction observed for HAdV2 stability evaluated by FC. In addition, the presence of organic matter in the surface freshwater might inhibit or minimize the adhesion process, maintaining the viruses in the aqueous phase due to the saturation of possible adhesion sites on the polypropylene container walls (Ward & Winston 1985; Gassilloud & Gantzer 2005).
The extended incubation times for PA and methods inherent to ICC-RT-qPCR may underestimate viral decay. PA requires six to seven days to allow a detectable cell-to-cell spread of viral progeny capable of producing sufficient cytopathic effect (CPE) for visual inspection. The macroscopic plaque formed due to this spread may arise from either a single virion or from a viral aggregate. Similarly, ICC-RT-qPCR is based on viral mRNA isolation from the entire cell monolayer followed by reverse transcription and qPCR; thereby allowing uncertainty regarding the number of virions which established the initial infection. Conversely, the use of labeled antibodies, short incubation times, and analysis of a single fluorescent (infected) cell in FC avoids these issues and more accurately assesses viral infectivity (Langlet et al. 2007; Hamza et al. 2011).
Studies evaluating the stability of enteric viruses usually employ a single assay to assess viral viability, which as demonstrated by this study, can vary depending on the selected method (Cromeans et al. 2010; Carratalà et al. 2013). To our knowledge, this is the first study that employed FC to evaluate the thermal stability of HAdV2. Rigotto et al. (2011) evaluated the stability of HAdV2 and 41 in sterilized surface water by PA and obtained a decay of 4 log10 after 301 days at 19 °C and no significant decay in samples stored at 4 °C. In the present work, when evaluated by PA, HAdV2 exhibited a decay of 4.64 log10 and 3.16 log10 after 230 days of storage at 22 and 4 °C.
Choosing to spike a non-sterile environmental water samples with viral stocks allowed a more real assessment of viral inactivation due to predation or virucidal activities of endogenous microorganisms, and explains more significant reductions in viral titers over time (Ward et al. 1986; Gordon & Toze 2003).
Thermal and temporal MNV-1 stability
Although samples kept at 22 °C exhibited the highest overall decay in viral titers, PA and RT-qPCR varied in inactivation rates. At 90 days post spiking, PA exhibited a 3.66 log10 reduction, while RT-qPCR peaked at 2.77 log10 after 180 days. When stored at −20 °C, the total log10 reduction was 2.53 log10 and 1.22 log10 for PA and RT-qPCR, respectively. At the end of 180 days, the samples stored at 4 °C showed a total log10 reduction values of 1.62 log10 and 1.36 log10 for PA and RT-qPCR, respectively, and 0.61 log10 and 0.70 log10 at −80 °C, respectively, for PA and RT-qPCR.
Despite the gradual reduction of the viral genome over time, this decay was lower than the observed for infectious MNV-1 particles. At 22 °C, MNV-1 infectivity showed the highest reduction values at 90 days after spiking, while reduction continued until the end of the study (180 days) at other temperatures. Bae & Schwab (2008) evaluated the stability of different HuNoV surrogates in environmental waters and suggested higher log reductions by PA compared with GC at 25 °C, but they found no differences at 4 °C. de Roda Husman et al. (2009), also obtained higher decay rates for RNA enteric viruses (PV1, PV2 and coxsackievirus B4) in artificial freshwater stored at 22 °C than at 4 °C evaluated by PA and RT-PCR, being the infectivity more affected than enterovirus genomes.
Other enteric viruses composed by RNA genomes, such as rotavirus (dsRNA), astrovirus and poliovirus (ssRNA) present different patterns of stability according to the water matrix. Specifically, infectivity remains less stable in surface water than groundwater; furthermore, ssRNA genome viruses are less stable than dsRNA virions (Charles et al. 2009; de Roda Husman et al. 2009).
There are few works evaluating viral stability under storage laboratory temperatures such as 4, −20 and −80 °C (Meng et al. 1987). Olson et al. (2004) showed that bacteriophage MS2, a surrogate for enteric viruses, exhibited lower stability when stored at −20 °C compared with 4 and −80 °C. The higher inactivation which occurred at −20 °C is probably due to the formation of crystals which can alter the viral capsid (Gould 1999).
Hurst et al. (1988b) evaluating the survival of different human enteroviruses (coxsackievirus B3, echovirus 7 and poliovirus 1), in different surface freshwaters stored at 22, 1 and −20 °C showed that the log10 values differed according to the virus and water type, and that some physicochemical parameters appeared to be more positively (conductivity) or negatively (turbidity and suspended solids) correlated with the virus survival in a specific storage temperature. In the present study, data regarding physicochemical parameters were obtained in a single sampling schedule being not possible the performance of a correlation test.
T90 values of HAdV2 and MNV-1 in surface freshwater
The T90 values of HAdV2 and MNV-1 were obtained through linear regression. The correlation values (R2), inactivation rate (−k) and the T90 are presented in Table 1.
|Virus .||Method .||Temperature (°C) .||R2 .||Inactivation rate (−k) .||T90 (days) .|
|Virus .||Method .||Temperature (°C) .||R2 .||Inactivation rate (−k) .||T90 (days) .|
*Linear regression between 30 to 230 days of evaluation.
†Linear regression between 10 to 230 days of evaluation.
‡Linear regression between 0 to 180 days of evaluation.
For HAdV2, T90 values varied between cell-culture based methods; specifically, PA T90 value was lower than FC T90. These differences, however, were not statistically significant (P > 0.05). PA analyses of samples stored at 22 and 4 °C exhibited lower T90 values than samples stored at −20 and −80 °C, which equated to 50 and 100 days, respectively. Conversely, FC and ICC-RT-qPCR assays exhibited nearly similar T90 values, regardless of storage temperature. However, samples stored at 4 °C and analyzed by FC exhibited the highest T90 values, being equivalent to 166 days (Table 1).
MNV-1 samples stored at 22 °C required less time to reach 1 log inactivation (33 days, Table 1) than other storage temperatures, but remained similar or higher relative to HAdV2 values obtained from PA (P > 0.05). MNV-1 PA exhibited classic linear regression throughout the 180 days experiment, regardless of storage temperature; however, HAdV2 linearity occurred from days 30 to 230 for PA and FC, and from days 10 to 230 for ICC-RT-qPCR.
Stability studies evaluating different enteric viruses in different environmental matrices, reported distinct patterns of persistence according to the virus type, inactivation treatments and methodologies (Espinosa et al. 2008; Cromeans et al. 2010; Verhaelen et al. 2012). In the present work, according to the titer reduction and T90 values, HAdV2 remained less stable than MNV-1 when stored at laboratory temperatures and assayed via PA. Furthermore, HAdV2 exhibited higher overall reduction in viral titers (log10), with T90 values similar or below MNV-1; however, this was not seen at 22 °C. The T90 values for MNV-1 and HAdV2 were 33 and 50 days, respectively. Despite the same water matrix and the same storage conditions, the comparison between both viruses is difficult since the T90 values were calculated at the start of linear decay, which occurred at varied sampling times.
In addition, the initial concentration of the viruses spiked in the freshwater samples may have an impact on the virus aggregation and can explain the apparent higher virus reduction for the HAdV2 in relation to the MNV-1 (Langlet et al. 2007).
In summary, the comparison between different the T90 inactivation values for enteric viruses or other fecal indicators, should take into account that many variables can contribute to the inactivation results, such as adhesion-aggregation phenomena, matrices and geographical sampling areas, variation assays, cell-culture or molecular methods employed, presence of endogenous microorganisms and statistical analysis (Noble et al. 2004; Ahmed et al. 2014).
This study evaluated the thermal and temporal stability of HAdV2 and MNV-1 in surface freshwaters stored at different temperatures. Samples stored at 22 °C demonstrated higher overall reduction in viral titers, suggesting a conjunction action of the temperature and endogenous micro-organism metabolism. When stored under laboratory storage temperatures, the decay of HAdV2 was higher than MNV-1. The stability of viruses under different environmental or laboratory conditions may vary between viruses and depends on the method utilized to assay infectivity. The findings from the presented work also suggest storing samples at lower temperatures for freshwater samples, at 4 °C for short- and at −80 °C for long-term periods, respectively.
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), project number 471755/2011-7 and scholarship from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES). We thank Dane C. Reano (University of California, Riverside) for reviewing the manuscript language.